Ella Samandar

Measurement of eight urinary metabolites of di(2-ethylhexyl) phthalate as biomarkers for human exposure assessment

Abstract Human metabolism of di(2-ethylhexyl) phthalate (DEHP) is complex and yields mono(2-ethylhexyl) phthalate (MEHP) and numerous oxidative metabolites. The oxidative metabolites, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono(2-carboxymethylhexyl) phthalate (MCMHP), have been considered to be better biomarkers for DEHP exposure assessment than MEHP because urinary levels of these metabolites are generally higher than MEHP, and their measurements are not subject to contamination. The urinary levels of the above metabolites, and of three other recently identified DEHP oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono-2-(1-oxoethylhexyl) phthalate (MOEHP), and mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), were measured in 129 adults. MECPP, MCMHP and MEHHP were present in all the samples analysed. MEHP and the other oxidative metabolites were detected less frequently: MEOHP (99%), MECBP (88%), MECPrP (84%), MEHP (83%) and MOEHP (77%). The levels of all DEHP metabolites were highly correlated (p<0.0001) with each other, confirming a common parent. The ω and ω-1 oxidative metabolites (MECPP, MCMHP, MEHHP and MEOHP) comprised 87.1% of all metabolites measured, and thus are most likely the best biomarkers for DEHP exposure assessment. The percentage of the unglucuronidated free form excreted in urine was higher for the ester linkage carboxylated DEHP metabolites compared with alcoholic and ketonic DEHP metabolites. The percentage of the unglucuronidated free form excreted in urine was higher for the DEHP metabolites with a carboxylated ester side-chain compared with alcoholic and ketonic metabolites. Further, differences were found between the DEHP metabolite profile between this adult population and that of six neonates exposed to high doses of DEHP through extensive medical treatment. In the neonates, MEHP represented 0.6% and MECPP 65.5% of the eight DEHP metabolites measured compared to 6.6% (MEHP) and 31.8% (MECPP) in the adults. Whether the observed differences reflect differences in route/duration of the exposure, age and/or health status of the individuals is presently unknown.

Environmental exposure to the plasticizer 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH) in US adults (2000—2012)

1,2-Cyclohexane dicarboxylic acid, diisononyl ester (DINCH) is a complex mixture of nine carbon branched-chain isomers. It has been used in Europe since 2002 as a plasticizer to replace phthalates such as di(2-ethylhexyl)phthalate (DEHP) and diisononyl phthalate (DINP). Urinary concentrations of the oxidative metabolites of DINCH, namely cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (MCOCH); cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester (MONCH); and cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (MHNCH), can potentially be used as DINCH exposure biomarkers. The concentrations of MCOCH, MONCH and MHNCH were measured by online solid phase extraction-high performance liquid chromatography-tandem mass spectrometry in urine collected in 2000 (n=114), 2001 (n=57), 2007 (n=23), 2009 (n=118), 2011 (n=94) and 2012 (n=121) from convenience groups of anonymous U.S. adult volunteers with no known DINCH exposure. None of the DINCH metabolites were detected in samples collected in 2000 and 2001. Only one sample collected in 2007 had measureable concentrations of DINCH metabolites. The detection rate for all three metabolites increased from 2007 to 2012. The presence of oxidative metabolites of DINCH in urine suggests that these oxidative metabolites can be used as DINCH biomarkers for exposure assessment even at environmental exposure levels.

Mono-(3-Carboxypropyl) Phthalate, A Metabolite of Di-n-Octyl Phthalate

Di-n-octyl phthalate (DnOP) is found as a component of mixed C6–C10 linear-chain phthalates used as plasticizers in various polyvinyl chloride applications, including flooring and carpet tiles. Following exposure and absorption, DnOP is metabolized to its hydrolytic monoester, mono-n-octyl phthalate (MnOP), and other oxidative products. The urinary levels of one of these oxidative metabolites, mono-(3-carboxypropyl) phthalate (MCPP), were about 560-fold higher than MnOP in Sprague-Dawley rats dosed with DnOP by gavage. Furthermore, MCPP was also found in the urine of rats dosed with di-isooctyl phthalate (DiOP), di-isononyl phthalate (DiNP), di-isodecyl phthalate (DiDP), di-(2-ethylhexyl) phthalate, and di-n-butyl phthalate (DBP), although at concentrations considerably lower than in rats given similar concentrations of DnOP. The comparatively much higher urinary concentrations of MCPP than of the hydrolytic monoesters of the high-molecular-weight phthalates DiOP, DiNP, and DiDP in the exposed rats suggest that these monoesters may be poor biomarkers of exposure to their precursor phthalates and may explain the relatively low frequency of detection of these monoester metabolites in human populations. MCPP and MnOP were also measured in 267 human urine samples. The frequent detection and higher urinary concentrations of MCPP than MnOP suggest that exposure to DnOP might be higher than previously thought based on the measurements of MnOP alone. However, because MCPP is also a minor metabolite of DBP and other phthalates in rats, and the metabolism of phthalates in rodents and humans may differ, additional data on the absorption, distribution, metabolism, and elimination of MCPP are needed to completely understand the extent of human exposure to DnOP from the urinary concentrations of MCPP.

Selecting Adequate Exposure Biomarkers of Diisononyl and Diisodecyl Phthalates: Data from the 2005–2006 National Health and Nutrition Examination Survey

Background High-molecular-weight phthalates, such as diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP), are used primarily as polyvinyl chloride plasticizers. Objectives We assessed exposure to DINP and DIDP in a representative sample of persons ≥ 6 years of age in the U.S. general population from the 2005–2006 National Health and Nutrition Examination Survey (NHANES). Methods We analyzed 2,548 urine samples by using online solid-phase extraction coupled to isotope dilution high-performance liquid chromatography–tandem mass spectrometry. Results We detected monocarboxyisooctyl phthalate (MCOP), a metabolite of DINP, and monocarboxyisononyl phthalate (MCNP), a metabolite of DIDP, in 95.2% and 89.9% of the samples, respectively. We detected monoisononyl phthalate (MNP), a minor metabolite of DINP, much less frequently (12.9%) and at concentration ranges (> 0.8 μg/L–148.1 μg/L) much lower than MCOP (> 0.7 μg/L– 4,961 μg/L). Adjusted geometric mean concentrations of MCOP and MCNP were significantly higher (p < 0.01) among children than among adolescents and adults. Conclusions The general U.S. population, including children, was exposed to DINP and DIDP. In previous NHANES cycles, the occurrence of human exposure to DINP by using MNP as the sole urinary biomarker has been underestimated, thus illustrating the importance of selecting the most adequate biomarkers for exposure assessment.

Temporal stability of eight phthalate metabolites and their glucuronide conjugates in human urine.

Humans are exposed to phthalates due to the ubiquitous use of these chemicals in consumer products. In the body, phthalates metabolize quickly to form hydrolytic and oxidative monoesters which, in turn, can be glucuronidated before urinary excretion. Exposure assessment studies typically report the total urinary concentrations of phthalate metabolites (i.e., free plus glucuronidated species). Nevertheless, because conjugation may potentially reduce the bioactivity of the metabolites by reducing their bioavailability, measuring the concentrations of free species may be of interest. An accurate, quantitative measurement of phthalate monoesters and their conjugated species requires data on the stability of these species in urine after sample collection and before analysis. We studied the stability of eight phthalate metabolites and their glucuronide conjugates at 25, 4, and -70 degrees C. Interestingly, the total concentrations of phthalate metabolites decreased over time at 25 and 4 degrees C, but not at -70 degrees C for up to 1 year and despite several freeze-thaw cycles. We further observed a considerable decrease in the concentrations of the glucuronides of some phthalate metabolites 1 day and 3 days after collection when the samples were stored at 25 and 4 degrees C, respectively. By contrast, the concentrations of the glucuronide conjugates at -70 degrees C remained unchanged for the whole duration of the study (1 year). Based on these findings, we recommend transferring urine specimens to a cooler or a refrigerator immediately after collection followed by permanent storage at subfreezing temperatures within hours of sample collection.

Identification of potential biomarkers of exposure to di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), an alternative for phthalate plasticizers

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is used as an alternative for some phthalate plasticizers. In rats, DINCH mostly eliminates in feces as cyclohexane-1,2-dicarboxylic acid (CHDA), mono isononyl ester (MINCH) or in urine as CHDA. However, CHDA is not a specific biomarker of DINCH and measuring MINCH in feces is impractical. To identify additional potential biomarkers, we administered DINCH (500 mg/kg body weight) in a single subcutaneous (SC) or oral dose to four adult female Sprague–Dawley rats. We collected 24-h urine samples before dosing (to be used as controls) and 24-h and 48-h after dosing, and serum at necropsy after 48 h. We positively identified and accurately quantified CHDA and cyclohexane-1,4-dicarboxylic acid, mono hydroxyisononyl ester (MHNCH) using authentic standards. Moreover, we tentatively identified MINCH and 12 oxidative metabolites, including 4 cyclohexane ring oxidation products, based on their mass spectrometric-fragmentation patterns. CHDA and MHNCH levels were higher in the urine collected 24 h after oral than SC administration. By contrast, 48-h after dosing, CHDA urinary levels were similar regardless of the exposure route. We detected all but two of the urine metabolites also in serum. Levels of CHDA and MHNCH in serum were lower than in the two post-dose urine collections. Our results suggest that several urinary oxidative metabolites, specifically CHDA, mono oxoisononyl ester and MHNCH may be used as specific biomarkers of DINCH exposure in humans.

Urinary and serum metabolites of di-n-pentyl phthalate in rats

Di-n-pentyl phthalate (DPP) is used mainly as a plasticizer in nitrocellulose. At high doses, DPP acts as a potent testicular toxicant in rats. We administered a single oral dose of 500 mg kg(-1)bw of DPP to adult female Sprague-Dawley rats (N=9) and collected 24-h urine samples 1d before and 24- and 48-h after DPP was administered to tentatively identify DPP metabolites that could be used as exposure biomarkers. At necropsy, 48 h after dosing, we also collected serum. The metabolites were extracted from urine or serum, resolved with high performance liquid chromatography, and detected by mass spectrometry. Two DPP metabolites, phthalic acid (PA) and mono(3-carboxypropyl) phthalate (MCPP), were identified by using authentic standards, whereas mono-n-pentyl phthalate (MPP), mono(4-oxopentyl) phthalate (MOPP), mono(4-hydroxypentyl) phthalate (MHPP), mono(4-carboxybutyl) phthalate (MCBP), mono(2-carboxyethyl) phthalate (MCEP), and mono-n-pentenyl phthalate (MPeP) were identified based on their full scan mass spectrometric fragmentation pattern. The ω-1 oxidation product, MHPP, was the predominant urinary metabolite of DPP. The median urinary concentrations (μg mL(-1)) of the metabolites in the first 24h urine collection after DPP administration were 993 (MHPP), 168 (MCBP), 0.2 (MCEP), 222 (MPP), 47 (MOPP), 26 (PA), 16 (MPeP), and 9 (MCPP); the concentrations of metabolites in the second 24 h urine collection after DPP administration were significantly lower than in the first collection. We identified some urinary metabolic products in the serum, but at much lower levels than in urine. Because of the similarities in metabolism of phthalates between rats and humans, based on our results and the fact that MHPP can only be formed from the metabolism of DPP, MHPP would be the most adequate DPP exposure biomarker for human exposure assessment. Nonetheless, based on the urinary levels of MHPP, our preliminary data suggest that human exposure to DPP in the United States is rather limited.

In Vitro Metabolites of Di-2-ethylhexyl Adipate (DEHA) as Biomarkers of Exposure in Human Biomonitoring Applications

Di-2-ethylhexyl adipate (DEHA) is a common plasticizer used in food packaging. At high doses, DEHA can cause adverse health effects in rats. Although the potential for human exposure to DEHA is high, no DEHA specific biomarkers are identified for human biomonitoring. Using human liver microsomes, we investigated the in vitro phase I metabolism of DEHA and its hydrolytic metabolite mono-2-ethylhexyl adipate (MEHA) and, for comparison purposes, of the analogous di-2-ethylhexyl phthalate (DEHP) and its hydrolytic metabolite mono-2-ethylhexyl phthalate. We unequivocally identified MEHA, a DEHA specific biomarker, and adipic acid, a nonspecific biomarker, using authentic standards. On the basis of their mass spectrometric fragmentation patterns, we tentatively identified two other DEHA specific metabolites: mono-2-ethylhydroxyhexyl adipate (MEHHA) and mono-2-ethyloxohexyl adipate (MEOHA), analogous to the oxidative metabolites of DEHP. Interestingly, although adipic acid was the major in vitro metabolite of DEHA, the analogous phthalic acid was not the major in vitro metabolite of DEHP. Our preliminary data for 144 adults with no known exposure to DEHA suggests that adipic acid is also the main in vivo urinary metabolite, while MEHA, MEHHA, and MEOHA are only minor metabolites. Therefore, the use of these specific metabolites for assessing the exposure of DEHA may be limited to highly exposed populations.

Erratum to: Exposure to di-2-ethylhexyl terephthalate in a convenience sample of U.S. adults from 2000 to 2016

DEHP metabolites (MECPP [45.5%], MEHHP [1.9%]) compared to the DEHTP metabolites (MECPTP [98.8%], MEHHTP [21.2%]). Contrary to the downward trend from 2000 to 2016 in urinary concentrations of MEHHP and MECPP, we observed an upward trend for MEHHTP and MECPTP. These preliminary data suggest that exposure to DEHTP may be on the rise. Nevertheless, general population exposure data using MEHHTP and MECPTP as exposure biomarkers would increase our understanding of exposure to DEHTP, one of the known DEHP alternatives.